Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
1999-12-09
2002-12-10
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S091100, C435S177000, C435S283100, C435S289100, C536S025400
Reexamination Certificate
active
06492162
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to a method for the recovery of nucleic acids from a nucleic acid-containing substance. In more detail, it relates to a method for the recovery of nucleic acids, which is suitable for an automatic device for the recovery of nucleic acid components as endogenous or exogenous genes from humor components for gene diagnosis by means of nucleic acid assays, or suitable for an automatic device for the recovery of plasmid DNAs from recombinant
E. coli
or the like for base sequencing of nucleic acids.
2. Description of the Related Art
A multitude of genetic technologies have been developed based upon advances in molecular biology, and a number of morbid genes have been isolated and identified according to these technologies. As a result, molecular biological techniques have been adopted as techniques for diagnosis or examination in the field of medical care so as to enable diagnosis which have been unable to conduct or to shorten the period of examination remarkably.
The significant advances largely owe to gene amplification techniques, in particular to polymerase chain reaction (hereinafter referred to as PCR: Saiki et al., Science, 239,487-491(1988)) techniques.
The PCR technique enables nucleic acids in a solution to amplify sequence-specifically so that it provides, for example, an indirect proof of the presence of a trace quantity of a virus in serum by amplifying and detecting nucleic acids as the viral gene.
The PCR technique is, however, somewhat disadvantageous in the use for clinical daily examinations. In particular, there are some difficulties in extraction and purification steps of nucleic acids in a pretreatment according to this technique, whereas the extraction and purification steps of nucleic acids have been indicated to be key steps (Ooshima et al., JJCLA 22(2),145-150(1997)).
These difficulties are attributed to inhibitory factors remaining in the purification step of nucleic acids, and known inhibitory factors include hemoglobin in blood, surfactant used in the extraction step and the like.
In addition, the extraction step requires a complicated procedure and a large amount of skilled labor. Therefore, this step is an obstacle to a new introduction of the genetic test into laboratories of hospitals, and the automatization of this step has been demanded.
On the contrary, plasmid DNAs are frequently used as materials for genetic engineering, and automatization of the extraction and purification steps of nucleic acids has been demanded from the viewpoint of labor savings in institutions for molecular biological research, as well as in the laboratories.
SUMMARY OF THE INVENTION
As a method for the recovery of nucleic acids from a biological sample at a high purity and being free from inhibitory factors, there has been known a method for the recovery of nucleic acids comprising the steps of allowing a surfactant to act on a biological sample in the presence of a protease to release nucleic acids, mixing the released nucleic acids with phenol (and chloroform), repeating aqueous phase (water phase)-organic phase isolation by a centrifuge several times and then recovering the nucleic acids as sediments from the aqueous phase with the use of an alcohol.
This method, however, has some disadvantages due to the use of an organic solvent such as phenol, a toxic substance, in the steps. To be more specific, an organic solvent such as phenol has the possibilities of dissolving plastics of a device used for the recovery of nucleic acids and of deteriorating the device.
Further, the method noted here requires for a centrifugation step to be effected. This leaves the problem that automation is made extremely difficult to achieve in putting and taking containers into and out of the rotators of a centrifuge and also in fractionating the resultant centrifuged solutions.
In addition, the use of an organic solvent such as phenol requires complicated treatments for discarding the organic solvent.
As other method for the recovery of nucleic acids than the method utilizing the aqueous phase-organic phase isolation, there have been reported a method for the recovery of DNAs from agarose gel utilizing the bonding properties of nucleic acids to the surface of glass in the presence of a chaotropic agent (B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. USA, 76(2),615-619(1979)), or a method for recovering plasmid DNAs from
E. coli
(M. A. Marko, R. Chipperfield and H. C. Birnboim, Anal. Biochem, 121,382-387(1982)).
A method for the recovery of nucleic acids from a biological sample by means of a simpler procedure is described in Japanese Unexamined Patent Publication No. 2-289596. According to this method, nucleic acids can be rapidly recovered by mixing a biological sample with a sufficient amount of a chaotropic agent (such as guanidinium salt) and silica beads and binding the free nucleic acids to a solid phase.
To obtain purified nucleic acids, however, this method requires a procedure for removing the guanidinium salt from the solid phase while retaining the nucleic acids bound to the solid phase, whereas a suitable removing procedure is not described.
For an efficient washing procedure of a solid phase at room temperature, the use of ethanol at least in a 75% concentration has been recommended in the past (C. W. Chen and C. A. Thomas, Jr., Anal. Biochem., 101,339-342(1980)). As ethanol is a volatile component, the mechanization of the method requires a close-open mechanism of a lid or a cooling mechanism for preventing ethanol from evaporation, and this invites upsizing of the device or deterioration of its reliability.
In addition, the method requires a washing step with 70% ethanol and/or acetone and hence requires a removing procedure of acetone by drying. When ethanol at a concentration of 70% or more and/or acetone, both of which are volatile, is mounted on an automatic device, the device requires a highly airtight chamber and a close-open mechanism of a lid and/or a cooling mechanism for preventing volatilization of the reagents, as described above.
The use of acetone has possibilities of dissolving plastics of a device employed for the recovery of nucleic acids and deteriorating the device due to its strong properties as an organic solvent, as mentioned above.
Therefore, materials of the device, cases and dispensers or other utensils to be used are remarkably limited. In addition, as acetone has acute toxicity and is inflammable, the release of acetone into the environment accompanied with the evaporation procedure should be avoided completely.
Japanese Unexamined Patent Publication No. 63-154696 discloses a method of isolating and purifying nucleic acids from an aqueous solution of a biological sample containing nucleic acids.
According to this method, an objective nucleic acid is recovered in the following manner: Initially, a biological sample is charged to a column filled with an anion exchanger, and the column is washed with an aqueous solution of sodium chloride to avoid the elution of an objective nucleic acid and to cleanse nonbinding components inclusive of carboxylated mucopolysaccharides. The objective nucleic acid is then recovered by eluting from the column with an aqueous solution of sodium chloride at such a concentration as to elute the objective nucleic acid.
The method just mentioned above ensures the recovery of an objective nucleic acid without using harmful substances.
This method where a sodium chloride aqueous solution is used as a washing solution, however, provides only a low recovery of nucleic acids. Accordingly, demands have been made to provide a method for recovering nucleic acids at a higher recovery yield.
To overcome the use of organic solvents in fractionating a nucleic acid, a method for the separation of nucleic acids is known in which a water-insoluble hybrid-forming carrier is supported on the surfaces of non-porous particles of 0.01-50 pm in particle diameter, the carrier having a single-chain nucleic acid bonded at least partly thereto (Japanese Une
Fukuzono Shinichi
Sakurai Toshinari
Yasuda Kenji
Mattingly Stanger & Malur, P.C.
Naff David M.
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